Biochemical characterization of a process that occurs at the membrane-cytosol interface is inherently challenging due to the bimodal nature of the reactants and products involved. A representative process potentially amenable to biochemical analysis resides in the biosynthesis of bacillosamine from the gram-negative enteropathogen Campylobacter jejuni. In C.jejuni bacillosamine is the first carbohydrate of a heptameric, prenylpyrophosphate-linked glycan that is assembled in the cytosol, transported across the membrane, and N-linked to nascent proteins in the periplasmic space. The locus that codes for enzymes involved in bacillosamine biosynthesis has been cloned and the heptameric glycan identified. However, the roles of the plasma membrane, transmembrane domains of the enzymes, and the membrane bound bactoprenolpyrophosphate molecule during glycan biosynthesis remains unclear. Outlined in this proposal are approaches designed to detail the biological mechanism of lipid anchored bacillosamine biosynthesis in model membranes. Full-length or separable domain constructs for each enzyme in the pathway will be heterologously expressed, purified, and respective enzymatic activity characterized in cell-free systems. Purified material will then be reconstituted in model membranes, and biochemical reactions characterized using lipid anchored substrate. The assays will also be used to identify the roles of the membrane, protein transmembrane domains, and the membrane bound prenylpyrophosphate molecule in bacillosamine biosynthesis. [unreadable] [unreadable]